Aggressive research in genomics, functional proteomics and drug discovery has resulted in a large increase in the number of chemical entities (“leads”) that have a potential for therapeutic activity. The leads are typically pruned in “pre-clinical screening” studies to select promising candidates for final “clinical studies.” Due to the large number of leads to be screened, the pre-clinical screening process has become a bottleneck in the drug discovery process.
During pre-clinical screening, sequential pharmacological transformations of the leads, in conjunction with an organism (e.g., cells, tissues, model animals, etc.,) are evaluated. The evaluation that is typically performed during pre-clinical screening is known as “ADMET” or sometimes “ADME Tox,” which is an acronym for Absorption, Distribution, Metabolism, Excretion and Toxicology. The absorption properties of leads are particularly important, and, as discussed later in this section, are particularly problematic to test.
There are generally two approaches to the pre-clinical screening of leads—in vivo testing and in vitro testing using artificial membranes (immobilized artificial membranes) or cell-based permeability methods. In vivo testing is performed within a living organism, while in vitro testing is performed outside of a living organism. Of these two approaches, in vivo testing provides a more accurate analysis of compound absorption and bio-availability during pre-clinical pharmaco-kinetic studies. Unfortunately, the logistics of animal-based studies makes them extremely expensive and time consuming. Furthermore, in vivo studies cannot provide the speed necessary to support high-throughput screening of drug candidates. Even the recently developed “cassette method,” wherein multiple compounds (about five to ten) are combined and administered to a single animal, cannot provide the desired productivity. (See, J. Berman et al., J. Med. Chem. 40:827–829 (1997); Dietz et al., U.S. Pat. No 5,989,918.)
Consequently, the focus in high-throughput screening of drug candidates is on various in vitro techniques (and even computer “in silico” modeling methods). Unfortunately, absorption is a difficult process to model and evaluate using in vitro testing. Specifically, absorption deals with the transport of compounds through live membranes (e.g., tissues, etc.)—a situation that is difficult to re-create outside of a living organism under test conditions. The successful application of in vitro models to the study of compound (e.g., drugs, nutrients, nutraceuticals, xenobiotics, etc.) transport across the intestinal mucosa therefore depends on how close those models imitate the characteristics of tissue in vivo.
A variety of in vitro methods have been developed to mimic the characteristic of the intestinal mucosa. One of the first methods that was developed for in vitro absorption studies is the everted sac technique (T. H. Wilson & G. Wiseman. J. Physiol. 123:116–125 (1954)).
In the everted sac technique, a segment of intestine (2–3 centimeters in length) is everted (i.e., turned inside out). The segment is filled with oxygenated buffer solution (serosal solution) and tied at both ends. The everted sac is placed in a similar buffer solution (mucosal solution) and incubated at body temperature with continuous oxygenation. The main reason for everting the segment is to ensure that the intestinal mucosa are adequately oxygenated, which is necessary for viability. The compound under study can be added to either the mucosal solution (on the outside) or the serosal solution (on the inside) depending on what kind of transport is being studied (mucosa→serosal or serosa→mucosal). After incubation is complete, the concentration of the transported compound is estimated in the serosal solution or on both sides of the intestine and in the intestinal mucosa.
This simple, reproducible, and inexpensive method is used for studying various mechanisms of compound uptake and transport through the intestine at its various regions as well as compound metabolism by intestinal mucosa (E. S. Foulkes. Proc. Soc. Exper. Biol. Med. 211:155–162 (1996)). The everted sac technique is also useful for studying the activity of the intestinal cell excretion system—P-glycoprotein—by comparing compound absorption in the presence and absence of P-glycoprotein inhibitors as well as in the presence of absorption enhancers and inhibitor screening (K. Hillgren et al., Med. Res. Rev. 15:83–109 (1995); L. Barthe et al., Fundam. Clin. Pharmacol. 13:154–168 (1999)).
Its utility notwithstanding, the everted sac technique has some disadvantages. One notable disadvantage is the animal origin of the intestine. Additional disadvantageous include low tissue viability and rapid histological damage in salt mixtures. Everted sacs look normal after inversion; however, studies have found that after 5 minutes of incubation at 37° C. morphological changes occur and after 30 minutes, 50–70% of epithelial cells were distorted (R. R. Levine et al., Eur. J. Pharm. 9:211–219 (1970)).
There are many modifications of everted sac technique. A recent modification proposed using tissue-cell medium instead of simple salt solutions for tissue incubation (L. Barthe et al., Europ. J. Drug Metab. Phatmacokinet. 23:313–323 (1998)). But these complex mixtures can be used in absorption studies only for a limited number of compounds.
Another serious drawback of the everted sac technique is that the serosal compartment is closed and cannot be properly oxygenated during incubation. While perhaps appropriate for short-term studies, the lack of proper oxygenation might cause problems for the evaluation of molecule kinetics during longer studies or when investigating compounds with a high absorption rate. Insufficient serosal oxygenation might also be a reason for low tissue viability because well-oxygenated intestinal tissue perfused with buffer solution remains viable for several hours (G. Parson & C. R. Paterson. J. Exper. Physiol. 50:220 (1965)).
Proper oxygenation of intestinal tissue from both the mucosal and serosal sides not only increases tissue viability but also affects compound absorption rate. In tests, everted sacs of rat intestine that have been filled with oxygen and incubated in oxygenated mucosal solution have exhibited a two-fold increase in glucose uptake compared with sacs that are oxygenated only from the mucosal surface (L. G. Ekkert & A. M. Ugolev. Gen. Physiol. Biophys. 2:141–157 (1982)).
Further drawbacks of the everted sac technique are that it is a manual technique and cannot support high-throughput compound screening or provide programmable operations with ease of setup and control functions.
A need remains for a technique and device that uses intestinal tissue for high-speed screening of compound absorption and that provides at least some of the following advantageous characteristics:    1. High-throughput screening (“HTS”) with low cost of preparation, operation and maintenance.    2. High-content screening (“HCS”), which enables multiple in-process sampling and testing that is necessary, for example, for kinetic studies.    3. Provides an ability to use live, un-everted intestinal segments as well as everted segments for absorption studies.    4. Provides an ability to use artificial membranes as necessary or desirable.    5. Preserves tissue viability and a high absorption rate due to bilateral oxygenation.    6. Preserves tissue viability by accelerating test procedures, including one or more of the tasks of loading, conditioning, and testing.    7. Provides an ability to use un-everted intestinal segments or artificial membranes for perfusion studies to imitate intestinal motility.    8. Maintains in vitro conditions that closely match in vivo conditions (e.g., pH, temperature, etc.) and the ability to monitor, control and alter these conditions.